Skin treatment system

ABSTRACT

A skin regeneration therapy combining precise bioelectric signals, light, and biologics for skin treatment and regeneration. Precise bioelectric signals give clear instructions to the stimulated cell DNA/RNA to produce specific regenerative proteins on demand. Bioelectric signals give clear instructions to cell membranes on what to let in and what to let out and serve as an equivalent or surrogate of environmental stimuli to cause a cell action in response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/129,533, filed Sep. 12, 2018, which will issue as U.S. Pat. No. 11,052,247 on Jul. 6, 2021, which is a continuation-in-part of U.S. patent application Ser. No. 15/812,760, filed on Nov. 14, 2017 and a continuation-in-part of U.S. patent application Ser. No. 15/460,129, filed on Mar. 15, 2017 (US 2017/0266371 A1, Sep. 21, 2017), which itself claims the benefit under 35 USC § 119 of:

U.S. Provisional Patent Application Ser. No. 62/308,702, filed Mar. 15, 2016;

U.S. Provisional Patent Application Ser. No. 62/363,012, filed Jul. 15, 2016;

U.S. Provisional Patent Application Ser. No. 62/364,472, filed Jul. 20, 2016;

U.S. Provisional Patent Application Ser. No. 62/375,271, filed Aug. 15, 2016;

U.S. Provisional Patent Application Ser. No. 62/385,124, filed Sep. 8, 2016;

U.S. Provisional Patent Application Ser. No. 62/454,521, filed Feb. 3, 2017; and

U.S. Provisional Patent Application Ser. No. 62/352,930, filed Jun. 21, 2016, the disclosure of each of which is incorporated herein in its entirety by this reference.

TECHNICAL FIELD

The application relates generally to the field of cosmetic and medical devices and associated methods and treatments, and more specifically to precise bioelectrical stimulation of a subject's skin tissue, augmented with the administration of a composition comprising, among other things, stem cells and nutrients, useful to stimulate and treat the subject, the subject's skin tissue(s) and/or cells.

BACKGROUND

Various organs and tissues of the body, such as skin, lose function due to aging. Other organs and tissues suffering from loss of function have been treated with electrical current to affect a change.

For example, U.S. Pat. No. 6,988,004 to Kanno and Sato (Jan. 17, 2006), the contents of which are incorporated herein by this reference, described a method for stimulating angiogenesis. The method comprised electrically stimulating muscle below the threshold for muscle contraction and increased VEGF mRNA.

For another example, see U.S. Pat. No. 7,483,749 (Jan. 27, 2009) to Leonhardt and Chachques, the contents of which are incorporated herein by this reference, describes a method for enhancing regeneration of the myocardium. The method comprised applying electrical stimulation to an injury site in the myocardium, and could be used in combination with implantation of myogenic cells into the injury site. The electrical stimulation could be applied before or after an implantation. Also described was that a bioelectric signal could recruit stem cells to the injury site.

BRIEF SUMMARY

Described is a skin regeneration therapy. The described therapy combines precise bioelectric signals, light, and biologics for skin treatment and regeneration. Precise bioelectric signals give clear instructions to the stimulated cell DNA/RNA to produce specific regenerative proteins. Bioelectric signals give clear instructions to cell membranes on what to let in and what to let out and serve as an equivalent or surrogate of environmental stimuli to cause a cell action in response.

In certain embodiments, described is a combination of bioelectrically induced stem cell homing, together with the controlled release and/or expression of tropoelastin, and, for example, a composition of mixed biological.

In certain embodiments, described is a combination of bioelectrically induced stem cell homing, proliferation, and differentiation, and the release and/or expression of tropoelastin.

Also described is bioelectric stimulator programmed to activate release in a subject's skin of, e.g., SDF-1, IGF-1, EGF, HGF, PDGF, eNOS, VEGF, Activin A and B, A, Follistatin, IL-6, HIF-1-α, and/or tropoelastin. Described is a bioelectric stimulator including: a power source (e.g., battery, capacitor, or other suitable source of electricity), and means for delivering an electrical signal to a subject's tissue (e.g., via electrode(s) or wirelessly). The bioelectric stimulator utilizes the electrical signal to precisely control protein expression in the tissue on demand. Such a bioelectric stimulator preferably precisely controls release of SDF-1 in the subject, without diminishing effect over time.

Also described is a method of using the bioelectric stimulator to regenerate and/or recover a subject's skin, the method including: delivering selected electrical signals to the skin so as to precisely control protein expressions in the right sequence and volume for skin regeneration and recovery.

Such a method can further include separately delivering to the subject a cocktail of regenerative agents. A preferred biological mix composition for such use includes (1) adipose-derived stromal vascular fraction (SVF), a mixture of growth factors including SDF1, IGF-1, IGF-1, PDGF, HGF, GDF10, and/or GDF11, (2) platelet rich fibrin (“PRF”) extended expression formulation, (3) amniotic fluid, (4) exosomes, (5) micro RNAs, (6) a nutrient hydrogel (e.g., LUMANAIRE™ hydrogel cream or other stem cell extract hydrogel based cream or gel), (7) alkaloids, (8) oxygenated nanoparticles, and (9) skin matrix.

Also described is a method of using the bioelectric stimulator in a subject's tissue to control release of a protein, wherein the electrical signal stimulates the production of a protein selected from the group consisting of SDF-1, IGF-1, HGF, EGF, PDGF, VEGF, HIF-1-α, eNOS, activin A, activin B, IL-6, follistatin, tropoelastin, and any combination thereof.

Also described is a method of using the bioelectric stimulator in a subject to repair DNA in the subject's skin, the method including: generating electrical signals from the bioelectric stimulator to control the release of IGF-1.

Also described is a method of using the bioelectric stimulator to achieve a desired result in a subject, wherein the desired result is skin regeneration or rejuvenation.

Also described is a bioelectric stimulator including: a power source (e.g., battery, capacitor, or other suitable source of electricity), and means for delivering an electrical signal to a subject's tissue (e.g., via electrode(s) or wirelessly), wherein the bioelectric stimulator utilizes the electrical signal to precisely control stem cell homing, proliferation and differentiation in the tissue. Such a bioelectric stimulator preferably utilizes the electrical signal to precisely control protein expression.

A preferred system includes:

1. A bioelectric stimulator that controls/stimulates the release/production of SDF-1, IGF-1, EGF, HGF, PDGF, eNOS, VEGF, Activin A and B, Follistatin, IL-6, HIF-1-α, and tropoelastin.

2. A micro infusion pump (e.g., a FLUIDSYNC™ micropump available from Fluidsynchrony of Pasadena, Calif., US), which is programmable and re-Tillable and preferably has a low cell damage design. Such a pump preferably includes a refilling silicon septum port or ports and reservoir chambers.

3. A multi-component composition that includes, for example, adipose-derived stem cells, muscle-derived stem cells (when needed for muscle), exosomes, Micro RNAs, nutrient hydrogel, growth factor cocktail, skin matrix, selected alkaloids, and/or selected anti-inflammatory agents.

The pump and stimulator may be associated with (e.g., connected to) the skin area to be treated/regenerated with a pacing infusion lead (available from Nanoscribe of Eggenstein-Leopoldshafen, Germany). The interface varies by the location of the skin, e.g., a conductive soft wrap can be used for certain applications.

The stimulator can be designed to externally deliver all regeneration promoting signals wirelessly to the subject's skin, associated tissue(s), and/or cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a programmed bioelectric stimulator together with a facemask and neck applicator. The facemask delivers bioelectric signals as well as LED light to the subject's face and neck.

FIG. 2 depicts a programmed bioelectric stimulator depicted alongside a U.S. quarter.

FIG. 3 depicts a micropump for use with the system.

FIG. 4 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing Activin B at 6.0 mV, pulse width 100 μs, square wave.

FIG. 5 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing EGF at 10V/cm (5V here), 500 Hz, pulse width 180 μs, square wave.

FIG. 6 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing follistatin at 10V/cm, 50 Hz, square wave.

FIG. 7 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing HGF at 3.5V, 10 second burst every 30 seconds, square wave.

FIG. 8 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing IGF-1: 3.0 mV, 22 Hz, square wave.

FIG. 9 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing PDGF30%: 3V/cm (100 mV here), 10 Hz, pulse width 200 μs, square wave.

FIG. 10 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing PDGF230%: 20V/cm (7.0V here), 100 Hz, pulse width 100 μs, square wave.

FIG. 11 depicts an image of the signal (voltage and frequency) associated with stem cell proliferation: 15 mV, 70 Hz, square wave.

FIG. 12 depicts an image of the signal (voltage and frequency) associated with stem cell proliferation: 2.5-6.0 V (4V here), 20 Hz, pulse width 200-700 μs, square wave.

FIG. 13 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing SDF-1: 3.5 mV, 30 Hz, square wave.

FIG. 14 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing tropoelastin: 60 mV, 50 Hz, square wave.

FIG. 15 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing VEGF: 100 mV, 50 Hz, square wave.

FIG. 16 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing SDF-1 (2^(nd) part): 0.25 mA (3.0V shown here), 100 Hz, 100 μs pulse width, square wave.

DETAILED DESCRIPTION

Referring now to FIG. 1, depicted is a human use stimulator and facemask for use with treatment of a subject's face and neck. As depicted in FIG. 2, the stimulator portion may be about the size of two quarters (available from QIG Greatbatch/Greatbatch, Inc. of Frisco, Tex., US) (FIG. 2). Depicted particularly in FIG. 1 are the face and neck mask (with straps), controller/stimulator, and carrying case.

In certain embodiments, the device provides bioelectric signaling sequences applied to the subject's skin are ones for (a) SDF-1 and/or PDGF (e.g., for stem cell homing to the treated area), (b) VEGF, PDGF, HIF-1-α, CXCL5, HGF, EGF, SDF1, and/or eNOS (e.g., for growing new blood vessels in the treated area), (c) tropoelastin (e.g., to increase the elasticity of skin in the treated area), (d) follistatin (e.g., to improve muscle tone in the treated area), and (e) IGF-1 (e.g., for DNA repair due to aging and sun damage in the treated area).

Preferably, a device provides bioelectric signaling sequences applied to the subject's skin are ones for (a) SDF-1 (stem cell homing), (b) tropoelastin (to turn back on the elasticity switch (“increase skin elasticity”) that turns off at age 9 in humans), (c) IGF-1 (for DNA repair), (d) VEGF, SDF-1, HGF, EGF, PDGF, eNOS, HIF-1-α, CXCL5, tropoelastin, and/or EGF (for dermal skin repair), (e) IL's (for inflammation response/inflammation management), (f) BMP proteins, and (g) Activin A and/or B.

In certain embodiments, a device provides bioelectric signaling sequences applied to the subject's skin are signals for: (a) SDF-1 (e.g., for stem cell homing to the treated area), (b) IGF-1 (e.g., for DNA repair due to aging and sun damage in the treated area), (c) tropoelastin (e.g., to increase the elasticity of skin in the treated area), and (d) VEGF (e.g., to improve blood circulation in the treated area). Preferably, such a device also provides bioelectric signaling sequences for application to the subject's skin for (e) PDGF, HIF-1-α, eNOS, and/or CXCL5, (e.g., to improve blood circulation in the treated area), (f) stem cell proliferation, (g) stem cell differentiation control, (h) extended PRF protein release, (i) HGF (e.g., to enhance skin regeneration), and/or (j) EGF (e.g., to aid or enhance skin regeneration).

The device may be similar in construction and form to the NUFACE® device of WO2006/116728 (Nov. 2, 2006), the contents of which are incorporated herein by this reference. The NUFACE® device comprises a hand-held housing from which a pair of electrodes project and circuitry for establishing a potential difference between the electrodes so that a microcurrent flows between the electrodes when the electrodes are placed on the skin. For other devices adaptable for use with the herein described system see, e.g., EP 0603451 A1 to Paolizzi (Jun. 29, 1994) and U.S. Pat. No. 8,639,361 to Nathanson (Jan. 28, 2014), the contents of each of which are incorporated herein by this reference. Similar devices are the LIGHTSTIM MULTIWAVE™ device for LED light therapy.

While such devices may be adapted for use herein, these prior art microcurrent devices were generally designed to accelerate healing via “current of injury” signaling, to improve mildly blood circulation and muscle tone and provide mild pain relief. For example, traditional TENS devices were designed to lower pain. Nearly all of these devices have relatively fuzzy/noisy signals compared to new modern precise bioelectric signaling stimulators, such as those used and programmed herein. Traditional microcurrent facial devices do not have specific, precise signals or sequences for controlling the release of specific regeneration promoting proteins on demand. Furthermore, even if they were programmed with these signals, they do not have the clarity of signal for the body to understand the instruction. Bioelectrical stimulators, such as those described herein, have precise programming to deliver precise clear signals to control protein expressions on demand. These controlled protein expressions are for very specific purposes.

In certain embodiments, the bioelectrical stimulation is provided by a SkinStim Model 240 High Precision Bioelectric and TENS Stimulator, which is pre-programmed for SDF-1, VEGF, IGF-1, and Tropoelastin Controlled Release. Bioelectric microcurrent and LED Face Mask (inner and outer views) such as a SkinStim Model 100 Micro-current and LED face mask may be used to treat the forehead, eyebrow, cheek, under-eye, jaw line, and jowls. Such a device preferably has, e.g., neoprene masks and straps (which are soft and oil and water resistant), a silicone outer casing of micro-current nodes and strap clasps, LED lights—rings that light up when mask is turned on, and metal nodes and wiring on inside of mask for micro-current.

Traditional microcurrent or TENS facials did not control with precision the release and/or expression of any of the above. At most, they provided a temporary, slight improvement of blood circulation. If there were however a surface wound, these general “current of injury” signals demonstrated accelerated healing.

In certain embodiments, a microcurrent and LED Mini-Mask Model 200 micro-current mini face mask is used. For applications just about the subject's eyes, a SkinStim EyeMask Model 100 microcurrent Eye mask may be used.

In certain embodiments, a pulsed laser light generator (e.g., one available from Epimedica of San Clemente, Calif., US) is used to provide laser light therapy to the area to be treated.

Methods and benefits of utilizing light and light emitting diodes (LEDs) for phototherapeutic treatment are described in U.S. Pat. No. 9,533,170 (Jan. 3, 2017) to Dye et al., U.S. Pat. No. 8,945,104 (Feb. 3, 2015) to Boone, III et al., and US 2006/0030908 A1 (Feb. 9, 2006) to Powell et al., the contents of each of which are incorporated herein by this reference.

Delivery may also/alternatively be through a micro-current facial conductive massage glove wherein, for example, electrodes associated with the bioelectrical stimulator are used to apply the desired electrical therapies.

Further, bioelectric signals may be used to improve muscle tone (follistatin for muscle tone improvement) and with improved muscle tone, the appearance of the overlying skin improves. Likewise, bioelectric signals may also be used to improve blood flow (VEGF, eNOS, PDGF, and HIF-1-αfor blood circulation improvement). IGF-1, EGF, HGF, Activin A+B, Follistatin and PDGF are expressed via bioelectric signaling and are intended to promote skin regeneration and DNA repair.

Typical subjects to be treated are humans, and the typical areas of skin are the face, neck, arms, the back of hands, legs, etc.

Skin regeneration compositions include basic skin regeneration compositions and advanced skin regeneration compositions. A basic skin regeneration composition contains, e.g., amniotic fluid and membranes, platelet rich fibrin (“PRF”) and PRF membranes, and nutrient engineered hydrogel. An advanced skin regeneration composition typically contains autologous (from patient to patient) and/or homologous stem cells (adipose-derived), ECM—matrix (skin matrix), micro RNAs, selected exosomes, selected alkaloids (e.g., tetraharmine), and oxygenated nanoparticles.

For instance, in certain embodiments, the skin regeneration composition contains bioelectric pre-treated stem cells (e.g., adipose tissue-derived), stromal fraction (“SVF”), PRF, selected growth factors, amniotic fluid, exosomes, micro RNAs in a gel, nutrient hydrogel, oxygenated nanoparticles, and skin matrix.

Stem cells may be obtained using a same-day stem cell process, which takes about 60 minutes. In such a process, first, one obtains tissue sample from the subject. Then a fat sample is processed using commercially available equipment and kits. This tissue is combined with reagent centrifuge and platelet rich fibrin (“PRF”). The stromal vascular fraction (“SVF”) is washed and filtered. Stem cells are re-suspended in saline or platelet rich plasma (“PRP”) and injected into the subject. The process may be repeated as needed or desired.

The stromal vascular fraction (SVF) of adipose tissue is a source of pre-adipocytes, mesenchymal stem cells (MSC), endothelial progenitor cell, T cells, B cells, mast cells as well as adipose tissue macrophages.

PRF may be provided by utilization of a SkinStim Bedside PRF Device or other platelet rich fibrin processing device.

This composition is preferably delivered repeatedly with a DERMAPEN™-like microneedle array over time. One such microneedle system is disclosed in US20170028184A1 to Godden et al. (Feb. 2, 2017) for a “Device and method of skin care and treatment via microneedles having inherent anode and cathode properties, with or without cosmetic or pharmacological compositions,” the contents of which are incorporated herein by this reference in its entirety.

A skin matrix is a composition comprising skin cells that are to be treated. The skin matrix is believed to aid in stem cell differentiation, but in any event is found to be useful in the composition. It has been found that for the multicomponent composition, cells plus selected growth factors are better than just cells alone. See, e.g., Procházka et al. “Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia,” Cell Transplantation, 25(9), pp. 1623-1633(11) (2016) and “Cocktail of Factors from Fat-derived Stem Cells Shows Promise for Critical Limb Ischemia,” world wide web at sciencenewsline.com/news/2016012204520017.html (Jan. 22, 2016), the contents of each of which are incorporated herein by this reference.

Useful hydrogels (and microRNA) are known and are described in Mao et al. “13—Hydrogel fibrous scaffolds for accelerated wound healing” Electrofluidodynamic Technologies (EFDTs) for Biomaterials and Medical Devices, pages 251-274 (2018), Bradshaw et al. “Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration” Nature Scientific Reports, vol. 4, Article number: 6903 (2014), Wang et al. “Local and sustained miRNA delivery from an injectable hydrogel promotes cardiomyocyte proliferation and functional regeneration after ischemic injury,” Nat Biomed Eng. 2017; 1: 983-992, doi: 10.1038/s41551-017-0157-y, R. Boyle “Wound-Treating Jelly Regenerates Fresh, Scar-Free Skin”, Popular Science, (Dec. 15, 2011), “New material developed for accelerated skin regeneration in major wounds,” Science Highlight, (National Institute of Biomedical Imaging and Bioengineering, Dec. 17, 2015), and Jouybar et al. “Enhanced Skin Regeneration by Herbal Extract-Coated Poly-L-Lactic Acid Nanofibrous Scaffold” Artif Organs. 2017 Nov; 41(11):E296-E307. doi: 10.1111/aor.12926.

Exosomes represent a specific subset of secreted membrane vesicles, which are relatively homogeneous in size (30-100 nm). Exosomes have been proposed to differ from other membrane vesicles by its size, density, and specific composition of lipids, proteins, and nucleic acids, which reflect its endocytic origin

Exosomes are formed in endosomal vesicles called multivesicular endosomes (MVEs) or multivesicular bodies, which originate by direct budding of the plasma membrane into early endosomes. The generation of exosomes to form MVEs involves the lateral segregation of cargo at the delimiting membrane of an endosome and inward budding and pinching of vesicles into the endosomal lumen. Because exosomes originate by two successive invaginations from the plasma membrane, its membrane orientation is similar to the plasma membrane. Exosomes from many cell types may contain similar surface proteins as the cell from which it is derived. Membrane proteins that are known to cluster into microdomains at the plasma membrane or at endosomes, such as tetraspanins (CD63, CD81, CD82), often are also enriched in EVs. It is also thought that endosomal sorting complex responsible for transport system and tetraspanins, which are highly enriched in MVEs, play a role in exosome production. How cytosolic constituents are recruited into exosomes is unclear but may involve the association of exosomal membrane proteins with chaperones, such as HSC70, that are found in exosomes from most cell types. MVEs are also sites of miRNA-loaded RNA-induced silencing complex accumulation, and the fact that exosome-like vesicles are considerably enriched in GW182 and AGO2 implicates the functional roles of these proteins in RNA sorting to exosomes. Exosomes are released to the extracellular fluid by fusion of MVE to the plasma membrane of a cell, resulting in bursts of exosome secretion. Several Rab GTPases such as Rab 27a and Rab27b, Rab11 and Rab35, all seem to be involved in exosomes release.

Useful exosomes are known and described in Hu et al. “Exosomes derived from human adipose mesenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts,” Nature Scientific Reports, vol. 6, Article number: 32993 (2016), Zhang et al. “Exosomes derived from human embryonic mesenchymal stem cells promote osteochondral regeneration,” Osteoarthritis and Cartilage, vol. 24, Issue 12, Dec. 2016,pp. 2135-2140, and Wu et al. “MSC-exosome: A novel cell-free therapy for cutaneous regeneration,” Cytotherapy, vol. 20, Issue 3, March 2018, pp. 291-301.

Generally, the system hereof involves a bioelectric stimulator controlling release of SDF-1, IGF-1, HGF, EGF, VEGF, PDGF, eNOS, follistatin, Activin A and B, and tropoelastin.

SDF-1 is generally for recruiting stem cells and maturing blood vessels. IGF-1 is for DNA repair. HGF is for tissue regeneration. EGF grows tissue. VEGF grows blood vessels. PDGF is a second stem cell homing factor and helps tissue regeneration. eNOS dilates blood vessels. Follistatin promotes muscle growth. Activin A and B regenerates nerve cells and neurons. Tropoelastin increases elasticity of all tissues especially the skin.

The micro voltage signal generator may be produced utilizing the same techniques to produce a standard heart pacemaker well known to a person of ordinary skill in the art. An exemplary microvoltage generator is available (for experimental purposes from Cal-X Stars Business Accelerator, Inc. DBA Leonhardt's Launchpads or Leonhardt Vineyards LLC DBA Leonhardt Ventures of Salt Lake City, Utah, US). The primary difference is the special electrical stimulation signals needed to control, e.g., precise follistatin release on demand (which signals are described later herein). The leading pacemaker manufacturers are Medtronic, Boston Scientific Guidant, Abbott St. Jude, BioTronik and Sorin Biomedica.

Construction of the electric signal generators and pacemakers, are known in the art and can be obtained from OEM suppliers as well as their accompanying chargers and programmers. The electric signal generators are programmed to produce specific signals to lead to specific protein expressions at precisely the right time for, e.g., optimal treatment or regeneration.

An infusion and electrode wide area pitch may be constructed by cutting conduction polymer to shape and forming plastic into a flat bag with outlet ports in strategic locations.

Micro stimulators may be purchased or constructed in the same manner heart pacemakers have been made since the 1960's. Micro infusion pumps can be purchased or produced similar to how they have been produced for drug, insulin, and pain medication delivery since the 1970's. The programming computer can be standard laptop computer. The programming wand customary to wireless programming wands may be used to program heart pacers.

Any one of the protein expression signals work well on their own, but they work better together. SDF-1 is the most powerful regeneration protein followed by IGF-1.

Wireless, single lumen infusion pacing lead or an infusion conduction wide array patch may all be used to deliver the regeneration signals and substances to the area of interest or they may be used in combination.

A re-charging wand for use herein is preferably similar to the pacemaker re-charging wand developed by Alfred Mann in the early 1970's for recharging externally implantable pacemakers.

A preferred composition includes adipose-derived cells (or bone marrow-derived MSCs or any pluripotent stem cell, such as iPS cells) and growth factor mix which should include (SDF-1, IGF-1, EGF, HGF, PDGF, VEGF, eNOS, activin A, activin B, follistatin, and tropoelastin plus selected exosomes (miR-146a, miR-294, mES-Exo) plus selected alkaloids (harmine and tetrahydroharmine) plus selected anti-inflammatory factors plus nutrient hydrogel (IGF-1, SDF-1, HGF plus FGF) plus skin matrix. Also, preferably included are amniotic fluid, placenta, or cord blood when available.

The concentration of cells in the compositions is preferably about 50,000,000 cells/ml. The amniotic fluid is preferably as described in Pierce et al. “Collection and characterization of amniotic fluid from scheduled C-section deliveries,” Cell Tissue Bank, DOI 10.1007/s10561-016-9572-7 (Springer, 2012) and is available from Irvine Scientific.

Described is a method of activating a tissue to differentiate a stem cell or to stimulate the tissue to produce a protein. The protein is selected from the group consisting of insulin-like growth factor 1 (“IGF-1”), epidermal growth factor (“EGF”), hepatocyte growth factor (“HGF”), platelet-derived growth factor (“PDGF”), endothelial NOS (“eNOS”), vascular endothelial growth factor (“VEGF”), activin A, activin B, follistatin, interleukin 6 (“IL-6”), hypoxia-inducible factor 1-alpha (“HIF-1-α”), and tropoelastin, the method including: stimulating the, e.g., human tissue with an electrical signal appropriate for the protein and tissue.

In such a method, when the electrical signal includes (within 15%): 0.1V applied at a frequency of about 50 Hz with a duration of about three (3) minutes (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is VEGF.

In such a method, when the electrical signal includes (within 2%): 200 picoamps for about 10 seconds for about one (1) hour and the pulse has an amplitude of about 5 volts and a width of about 0.5 milliseconds for about 1 hour, with a duration of about one (1) minute (wherein the electrical signal is as measured three (3) mm deep into the tissue), stem cells differentiate.

In such a method, when the electrical signal includes (within 15%): 10V at 50 HZ and 100 HZ for about 12 hours each (duration 1 minute) (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is follistatin.

In such a method, when the electrical signal includes (within 15%): 3.5V stimulation in 10 second bursts, 1 burst every 30 seconds at a frequency of about 50 HZ (duration 5 minutes) (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is HGF.

In such a method, when the electrical signal includes (within 15%): 3 mv with a frequency of about 22 Hz, and a current of about 1 mA for about fifteen (15) minutes and 3 ma for about fifteen (15) minutes (duration 5 minutes) (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is IGF-1.

In such a method, when the electrical signal includes (within 15%): 0.06 V with 50 Z alternating electrical field and a current of about 1 ma for about fifteen (15) minutes and 3 ma for about fifteen (15) minutes (duration 2 minutes) (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is tropoelastin.

In such a method, when the electrical signal includes (within 15%): alternating high-frequency (HF) and medium-frequency signals (MF), symmetric, biphasic, trapezoid pulses, with 400-μs pulse duration and 1.5/1-s ramp-up/ramp-down duration, respectively (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is eNOS. In such a method, when the HF consists of about 75 Hz pulses with six (6) seconds on and 21 seconds off for about fifteen (15) minutes. In such a method, when the MF consists of about 45 Hz pulses with 5 seconds on 12 seconds off for about fifteen (15) minutes followed by stimulation duration set as 20 minutes. In such a method, when the electrical signal includes (within 15%): 1 Hz stimulation, stimulation applied for about nine (9) seconds, followed by a one (1) second silent period, a total of about 1080 stimulations for about 20 minutes. In such a method, when the electrical signal includes (within 15%): 20 Hz stimulation, stimulation applied for about two (2) seconds, followed by silent period for about 28 seconds, a total of about 1600 stimulations for about 20 minutes (duration 2 minutes).

In such a method, when the electrical signal includes (within 15%): 6 mv at 150 HZ Monophasic square wave pulse 0.1 ms in duration current of fifteen (15) mA for about fifteen (15) minutes (duration two (2) minutes) (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is Activin B.

In such a method, when the electrical signal includes (within 15%): 10 V/cm, pulse-width 180 μs, 500 Hz (duration nine (9) minutes) (wherein the electrical signal is as measured three (3) mm deep into the tissue), the protein produced is EGF.

For example, upregulation of IGF-1, VEGF, and SDF-1 was achieved in cardiomyocytes using such signals. Upregulation of SDF-1 was achieved in pig heart. Upregulation of VEGF, endothelial NOS (“eNOS”), hypoxia-inducible factor 1-alpha (“HIF-1-α”), and IL-6 was achieved in eye cells.

Also described is a method of activating a tissue to produce stromal cell-derived factor 1 (“SDF-1”), the method including: stimulating the (e.g., human) tissue with an electrical signal, wherein the electrical signal includes (within 15%): 30 pulses per second with a voltage of about 3.5 mV, and successively alternating currents of about 700 to 1500 picoamps for about one minute, and again with 700 to 1500 picoamps for about one minute and stimulated with current of about 0.25 mA, pulse duration of about 40 pulses/s, pulse width of about 100 μs, wherein the electrical signal is as measured three (3) mm deep into the tissue (e.g., preferably for a period of time of about 20 minutes).

Further described is a method of activating a tissue to attract a stem cell, the method including: stimulating the (e.g., human) tissue with an electrical signal, wherein the electrical signal includes (within 2%): fifteen (15) mV and a current of about 500 picoamps at 70 pulses per minute for about three (3) hours and 20 pulses per minute, a pulse amplitude of from about 2.5-6 volts, and a pulse width of from about 0.2-0.7 milliseconds for about three (3) hours for about three (3) minutes, wherein the electrical signal is as measured three (3) mm deep into the tissue.

In some cases, SDF-1 recruits via a presumed homing signal new reparative stem cells to the damaged skin. VEGF causes new nutrient and oxygen producing blood vessels to grow into the area being treated. IGF-1 repairs damaged cells and tissues. Follistatin repairs damaged muscle. Tropoelastin adds elasticity to treated tissues making them more compliant. HGF aides in all repair processes. All of these proteins work together to fully regenerate/rejuvenate the skin tissue over time.

The healing process can be accelerated with the use of a micro infusion pump that is filled with various types of stem cells and growth factors and in some cases drugs.

What follows are preferred signals from the stimulator. For example, described are two PDGF expression control signals, one low voltage and one higher voltage. The test tissue is sheep heart tissue. The test cells are mesenchymal stem cells.

30% PDGF increase: 3 V/cm, 10 Hz, 2 micro amps (0.000002 amps) and the pulse duration 0.2 ms.

230% PDGF increase: 20 V/cm 100 Hz, 0.25mA (2.5e-7 amps) and pulse duration of 40 pulses/s, width of 100 μs.

40 minute treatment cycles 2 times a week for 4 weeks and then 3 times a week for 12 weeks.

PDGF Signal: 20V for 1 minute, 20 mV for 10 minutes, current of 0.25 mA, pulse duration of 40 pulses/s, pulse width of 100 μs, and frequency of 100 Hz for 5 minutes followed by 528 Hz for 3 minutes and 432 Hz for 3 minutes and 50 Hz for 3 minutes.

VEGF—Blood vessel sprouting growth: 0.1V applied at a frequency of 50 Hz. Duration 3 minutes. In certain embodiments, the duration can be for a time of, e.g., from 10 to 40 minutes, wherein the percentage VEGF expression increases with time.

SDF-1—Stem cell recruiting signal: 30 pulses per second with a voltage of 3.5 mV, and successively alternating currents of 700 to 1500 picoamps for one minute, and again with 700 to 1500 picoamps for one minute and stimulated with current of 0.25 mA, pulse duration of 40 pulses/s, pulse width of 100 μs, and frequency of 100 Hz—each signal for 40 minutes to 8 hours a day for 2 to 36 months as needed for ideal results. Duration 7 minutes.

Stem cell proliferation signals: 15 mV and a current of 500 picoamps at 70 pulses per minute for 3 hours and 20 pulses per minute, a pulse amplitude of from 2.5-6 volts, and a pulse width of from 0.2-0.7 milliseconds for 3 hours. Duration 3 minutes.

Stem cell differentiation signals to become muscle: 200 picoamps for 10 seconds for 1 hour and the pulse has an amplitude of 5 volts and a width of 0.5 milliseconds for 1 hour. Duration 1 minute. Another method is to reverse polarity and drop the voltage.

Stem cell differentiation signal to become skin: low-voltage square wave with 60 ms pulse duration for one to seven cycles, then reverse polarity to a negative square wave for one to fourteen cycles, which repeats, delivering 200 microAmps.

Follistatin—(muscle growth) production signal: 10V at 50 HZ and 100 HZ 0.25mA. Duration 1 minute.

HGF—Hepatocyte growth factor (arrhythmia reduction) signal: 3.5V stimulation in 10 second bursts, 1 burst every 30 seconds at frequency 50 HZ. Duration 5 minutes.

IGF-1: 3mv with electric frequency of 22 Hz, and electric current of 1 mA for 15 minutes and 3ma for 15 minutes. Duration 5 minutes.

Tropoelastin: 0.06 V with 50 Z alternating electrical field and electric current of 1 ma for 15 minutes and 3 ma for 15 minutes. Duration 2 minutes.

eNOS: Alternating high-frequency (HF) and medium-frequency signals (MF): Symmetric, biphasic, trapezoid pulses, with 400-μs pulse duration and 1.5/1-s ramp-up/ramp-down duration, respectively. HF consisted of 75 Hz pulses with 6 second on-21 second off for 15 minutes. MF consisted of 45 Hz pulses with 5 second on-12 second off for 15 minutes. Followed by stimulation duration set as 20 minutes for both 1 Hz and 20 Hz stimulations. For 1 Hz stimulation, stimulation is applied for 9 seconds, followed by a 1 second silent period, a total of 1080 stimulations for 20 min. For 20 Hz stimulation, stimulation is applied for 2 seconds, followed by silent period for 28 seconds, a total of 1600 stimulations for 20 min. Duration 2 minutes.

Activin B: 6 mv at 150 HZ Monophasic square wave pulse 0.1 ms in duration current of 15 mA for 15 minutes. Duration 2 minutes.

EGF—10 V/cm, pulse-width 180 μs, 500 Hz. Duration 9 minutes.

FIGS. 4-14 are images of the corresponding signals with the name, voltage, and frequency of each signal written on each image. eNOS and differentiation signals were omitted due to of complexity or lack of frequency parameters. The signals are to be further defined in terms of current and frequency, not voltage and frequency as shown. The voltage delivered to the cells will be different for each tissue type, but with current all of the signals can be kept constant regardless of tissue type. The device should have a current driven signal (instead of voltage driven like most other devices).

Specifically, FIG. 4 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing Activin B at 6.0 mV, pulse width 100 μs, square wave on a TEKTRONIX® TPS 2024 four channel digital storage oscilloscope. FIG. 5 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing EGF at 10V/cm (5V here), 500 Hz, pulse width 180 μs, square wave. FIG. 6 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing follistatin at 10V/cm, 50 Hz, square wave. FIG. 7 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing HGF at 3.5V, 10 second burst every 30 seconds, square wave. FIG. 8 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing IGF-1: 3.0 mV, 22 Hz, square wave (for a time of, e.g., from 10 to 40 minutes). FIG. 9 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing PDGF30%: 3V/cm (100 mV here), 10 Hz, pulse width 200 μs, square wave. FIG. 10 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing PDGF230%: 20V/cm (7.0V here), 100 Hz, pulse width 100 μs, square wave. FIG. 11 depicts an image of the signal (voltage and frequency) associated with stem cell proliferation: 15 mV, 70 Hz, square wave. FIG. 12 depicts an image of the signal (voltage and frequency) associated with stem cell proliferation: 2.5-6.0 V (4V here), 20 Hz, pulse width 200-700 μs, square wave. FIG. 13 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing SDF-1: 3.5 mV, 30 Hz, square wave. FIG. 14 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing tropoelastin: 60 mV, 50 Hz, square wave. FIG. 15 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing VEGF: 100 mV, 50 Hz, square wave. FIG. 16 depicts an image of the signal (voltage and frequency) associated with producing and/or expressing SDF-1 (2nd part): 0.25 mA (3.0V shown here), 100 Hz, 100 μs pulse width, square wave.

In certain embodiments, a subject's skin is first scanned or analyzed with a device to determine what his or her needs may be before treatment begins. The scanning/analysis can be by, e.g., generating mechanical vibrations at position adjacent the location to be an analyzed as described in, e.g., US 2003/0220556 A1 to Porat et al. (the contents of which are incorporated herein by this reference) and/or by measuring transmembrane voltage potential of a cell (see, e.g., Chernet & Levin, “Transmembrane voltage potential is an essential cellular parameter for the detection and control of tumor development in a Xenopus model,” Dis. Models & Mech. 6, pp. 595-607 (2013); doi:10.1242/dmm.010835), the contents of which are also incorporated herein by this reference. See, also, Brooks et al. “Bioelectric impedance predicts total body water, blood pressure, and heart rate during hemodialysis in children and adolescents” J. Ren. Nutr., 18(3):304-311 (May 2008); doi: 10.1053/j.jrn.2007.11.008, the contents of which are incorporated herein by this reference, describing the use of bioelectric impedance to evaluate the variability of blood pressure, systolic blood pressure, etc.

As used herein, “scanning” means measuring bioelectrical electrical activity of skin, sometimes by placement of a bion coil reader and transmitter in the skin, and direct that information to a computer. The computer stores the bioelectrical read measurements of diseased skin and healthy skin and makes a comparative exam classifying the skin into one category or another, which is much like a doctor using information to make a diagnosis.

Scanners such as the Ina'Chi scanner, the Quantum Magnetic Resonance Analyzer (QMRA), the 3D Quantum Health Analyzer Scan whole body organ health 2, BODYSCAN® scanner, and the “BIONic muscle spindle” are also useful.

In an alternative embodiment, the analysis conducted by the device comprises (or further includes) detecting minute energy fields around the human body with, e.g., a “SQUID magnetometer” (SQUID is an acronym for “Superconducting Quantum Interference Device”), able to detect biomagnetic fields associated with physiological activities in the subject's body. A quantum resonant magnetic analyzer analyzes such fields. The magnetic frequency and energy of a subject's tissue(s) are collected by appropriately positioning the sensor with respect to the portion of the subject's tissue(s) to be analyzed, and after amplification of the signal by the instrument, the data are compared with standard quantum resonant spectrum of diseases, nutrition, and other indicators/markers to determine whether the sample waveforms are irregular using a Fourier approach.

In certain embodiments, bioelectric signaling is applied to the area of skin to be treated in approximate 28 minute treatment sessions twice a week for, e.g., up to 16 weeks (32 times total) utilizing, e.g., a benchtop bioelectric stimulator and face mask. The bioelectric signaling is preferably applied to the skin area to be treated as follows: (a) SDF-1 homing signal to recruit stem cells to skin for about seven (7) minutes, (b) IGF-1 DNA repair signal for about four (4) minutes, (c) tropoelastin signal to increase skin elasticity for about twelve (12) minutes, and (d) blood circulation improvement signal sequence VEGF for about five (5) minutes.

This “basic” program can be supplemented by supplying further signaling (i.e., in addition to the foregoing) by applying the following bioelectric signaling: (e) PDGF, HIF1a, eNOS, CXCL5 for advanced blood circulation, (f) Stem cell proliferation, (g) Stem cell differentiation control, (h) extended PRF protein release, (i) HGF for skin regeneration, and (j) EGF for skin regeneration.

A preferred treatment protocol for facial skin regeneration, rejuvenation, and/or treatment comprises: 30 minutes of bioelectric treatments (e.g., in clinic), twice a week for 16 weeks; PRF, amniotic fluid, stem cell injections (via, e.g., DERMAPEN™) once a week every four weeks for 16 weeks (four times total); amniotic fluid membrane application once a week every eight weeks for 16 weeks (two times total); daily bioelectric treatment (e.g., at home) for at least 15 minutes a day for 16 weeks; bioelectric micro-current conductive globe facial massage once a week every four weeks for 16 weeks (four times total); electroacupuncture once a week every eight weeks for 16 weeks (two times total); and LUMANAIRE™ hydrogel skin cream applied morning and night every day for 16 weeks.

A preferred protocol follows. First, bioelectric signaling is applied to the area to be treated in approximate 40 minute treatment sessions twice a week for up to 16 weeks (32 times total) utilizing, e.g., a benchtop bioelectric stimulator and face mask. The preferably in-clinic precision bioelectric signaling applied to the area is as follows:

(first) SDF-1 homing signal to recruit stem cells to skin for about seven (7) minutes,

(second) stem cell to skin differentiation signal for about three (3) minutes,

(third) IGF-1 DNA repair signal for about four (4) minutes,

(fourth) EGF epidermal growth factor signal skin repair for about three (3) minutes,

(fifth) Tropoelastin signal to increase skin elasticity for about twelve (12) minutes,

(sixth) Blood circulation improvement signal sequences VEGF, PDGF, eNOS, HIF1a, CXCL5, EGF, HGF, and SDF-1 for about five (5) minutes,

(seventh) Muscle-toning signal follistatin for about two (2) minutes,

(eighth) SDF-1 again for about one (1) minute, and

(ninth) Stem cell to skin differentiation again for about one (1) minute.

Then, the foregoing bioelectric signaling is preferably combined with any or all of the following:

DERMAPEN™ Micro Needle array delivery of a skin regeneration composition mix that includes adipose tissue derived stem cells, exosomes, micro RNAs, selected alkaloids, hydrogel skin matrix, elastin, oxygenated nanoparticles, platelet rich fibrin (“PRF”), amniotic fluid, and selected growth factors such as SDF-1, IGF-1, EGF, HGF, and PDGF or any combination thereof once a month for about four (4) months (four times total)

DERMAPEN™ micro needle array delivery of PRF once a month for about four (4) months (four times total). May or may not be bioelectric energy enhanced.

DERMAPEN™ micro needle array delivery of amniotic fluid once a month for about four (4) months (four times total)

DERMAPEN™ micro needle array delivery of adipose tissue derived stem cells once every two months for about four (4) months (two times total)

At home Prizm Medical electrical stimulation with conductive electro-massaging gloves once a week for 15 minutes for 16 weeks (sixteen times total)

Electroacupuncture with, e.g., simple electroacupuncture pen once a month for about four (4) months (four times total)

LED pulsed light therapy 10 minutes twice a week via combination bioelectric and light mask (32 times total)

Amniotic fluid membrane dressings once a month left on for one hour (four times total).

DERMAPEN™ micro needle array delivery of oxygenated nanoparticles once a month for four months (four times total)

DERMAPEN™ micro needle array delivery of hydrogel skin matrix once every other month for four months (two times total)

Application of a hydrogel and stem cell matrix-based skin cream twice a day for about sixteen (16) weeks once in the morning and once before bed (224 times total). May or may not be light or bioelectric energy activated or enhanced.

Used in conjunction with GOSEAR™ electroacupuncture pen, DERMAPEN™ microneedle array for delivering stem cells, amniotic fluid, PRF. PRF bedside processing device plus bioelectric PRF equals “BPRF.”

The invention is further described with the aid of the following illustrative Examples.

Examples

The study is to enroll and treat patients to assess improvement in the appearance of facial wrinkles utilizing a bioelectric or biologics (PRF and amniotic fluid) therapy or a combination of bioelectric and biologics therapy. The study is to enroll and treat a minimum of 49 subjects (23 in treatment Group A receiving a bioelectric treatment alone, 23 in treatment Group B receiving biologics treatment alone and 23 in Group C receiving combined bioelectric and biologics treatment) with moderate facial wrinkles corresponding to a grade of 4-6 on the validated Fitzpatrick Wrinkle Assessment Scale.

Group A=Active Comparator: Bioelectric Treatment Alone (treatment of facial wrinkle(s) with bioelectric treatment only and hydrogel skin cream). Devices: SkinStim Bioelectric Stimulation twice a week for 30 minutes for 12 weeks and once a week 20 minutes electro face massages with Prizm Medical stimulator and conductive gloves and hydrogel skin cream applied twice a day morning and evening.

Group B=Active Comparator: Biologics Treatment Alone (treatment of facial wrinkle with PRF and amniotic fluid both delivered via a DERMAPEN™ micro needle array and hydrogel skin cream comparison of bioelectric versus biologics versus combined bioelectric and biologics therapies).

Group C: Active Comparator: Combined Bioelectric and Biologics Treatment (treatment of facial wrinkle with bioelectric and biologic treatments) Devices: SkinStim Stimulation twice a week for 30 minutes for 12 weeks and once a week 20 minute electro face massages with Prizm Medical stimulator and conductive gloves and Biologics: Autologous PRF and amniotic fluid applied via DERMAPEN™ microneedle array delivery once a month for 3 months and hydrogel skin cream applied twice a day morning and evening comparison of bioelectric versus biologics versus combined bioelectric and biologics therapies

Primary Outcome Measure:

1. Fitzpatrick Wrinkle Assessment [Time Frame: change in Fitzpatrick Wrinkle Score between baseline and 90 days post treatment assessment.]

Subject photos will be evaluated using the 9-point Fitzpatrick Wrinkle Assessment Scale at all follow up visits. An improvement is noted by a decrease in the numeric Fitzpatrick Wrinkle score. The Fitzpatrick Wrinkle Assessment ranges from 1-9. Wrinkle Score between baseline and 90 days post treatment assessment. Positive values indicate an increase in score, while negative values indicate a decrease.

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1. (canceled).
 2. A method of treating an area of a subject's skin, the method comprising: applying the following bioelectric signals to the area of the subject's skin: (a) a first bioelectric signal that upregulates expression of stromal cell-derived factor 1 (SDF1) at the treated area; (b) a second bioelectric signal that upregulates expression of platelet-derived growth factor (PDGF) at the treated area; (c) a third bioelectric signal that upregulates expression of tropoelastin at the treated area; (d) a fourth bioelectric signal that upregulates expression of follistatin at the treated area; and (e) a fifth bioelectric signal that upregulates expression of insulin-like growth factor 1 (IGF-1) at the treated area.
 3. The method according to claim 2, further comprising: delivering to the area of skin a skin regeneration composition comprises bioelectric pre-treated stem cells, bioelectric pre-treated adipose tissue-derived stem cells, stromal fraction (SVF), platelet rich fibrin (PRF), growth factors, amniotic fluid, exosomes, micro RNAs in a gel, nutrient hydrogel, oxygenated nanoparticles, and extracellular matrix (ECM).
 4. The method according to claim 2, further comprising: delivering platelet rich fibrin (PRF) to the area of the subject's skin.
 5. The method according to claim 4, wherein the PRF is delivered via a microneedle system.
 6. The method according to claim 2, further comprising: delivering amniotic fluid to the area of the subject's skin.
 7. The method according to claim 6, wherein the amniotic fluid is delivered via a microneedle.
 8. The method according to claim 2, further comprising: suspending stem cells in platelet rich plasma (PRP); and injecting the thus suspended stem cells into the subject.
 9. The method according to claim 2, further comprising: delivering light emitting diode (LED) pulsed light therapy to the area of the subject's skin.
 10. The method according to claim 2, further comprising: delivering exosomes to the area of the subject's skin.
 11. The method according to claim 2, further comprising: delivering to the area of skin a skin regeneration composition comprising amniotic fluid, amniotic fluid membranes, platelet rich fibrin (PRF), PRF membranes, and nutrient hydrogel.
 12. The method according to claim 2, wherein the first bioelectric signal has (within 15%) a frequency of 30 Hz, square wave.
 13. The method according to claim 2, wherein the first bioelectric signal has a frequency of 100 Hz, square wave.
 14. The method according to claim 2, wherein the second bioelectric signal has a frequency of 10 Hz, square wave.
 15. The method according to claim 2, wherein the second bioelectric signal has a frequency of 100 Hz, square wave.
 16. The method according to claim 2, wherein the third bioelectric signal has (within 15%) a frequency of 50 Hz, square wave.
 17. The method according to claim 2, wherein the fourth bioelectric signal has (within 15%) a frequency of 50 Hz, square wave.
 18. The method according to claim 2, wherein the fifth bioelectric signal has (within 15%) a frequency of 22 Hz, square wave. 